Evaluation of High Content Imaging Technology and Associated Methods for the Acceleration of Cell-based Assay Development

Transcription

1 Evaluation of High Content Imaging Technology and Associated Methods for the Acceleration of Cell-based Assay Development Heather Ann Brauer Potency Assay Group

2 Outline Introduction o Seattle Genetics o Cell-based assay development State of technology Applications for cell-based assay development o Cell line characterization o Early assay development o Plate effect assessment o Failed sample evaluation Conclusions

3 Seattle Genetics: Demonstrating Broad Progress A biotechnology company focused on developing and commercializing empowered antibody-based therapies for the treatment of cancer o Industry leader in next-generation antibody-drug conjugates (ADCs) ADCETRIS (brentuximab vedotin) o o Approved in 60 countries Broad clinical development program in CD30-expressing lymphomas Vadastuximab talirine (SGN-CD33A) o Expected to advance into phase 3 pivotal trial for AML in 2016 More than a dozen clinical and preclinical pipeline programs Company facts o Founded in 1998 o o o Headquartered in Bothell, WA Publicly traded (Nasdaq: SGEN) More than 700 employees

4 ADC Technology: Empowering Antibodies ADCs combine the targeting ability of monoclonal antibodies with the potency of cytotoxic agents Designed to improve efficacy and reduce toxicity Potent cytotoxic agents and stable linkers with long half-lives Readily scalable through simple, reproducible synthesis SGEN technology empowers more than 25 of the ADCs in clinical development across the industry, including both proprietary and collaborator programs

5 Introduction Biological understanding at the level of the cell is critical for assay performance. Multi-faceted approaches can be used for early development, robustness studies, and sample evaluation. Acceleration of cell-based bioassay development is possible with new technologies.

6 State of Technology Composite Plate Reader FACS & Laser Scanning Confocal High-Content Real- Time Cell Imaging High-Content Subcellular Imaging Spinning Disc Confocal

7 Live-Cell Based Imaging Platform Real-time quantitative live-cell based imaging and analysis Kinetic data collection Cell incubator based microscope o Collect and process image data Various objectives (4x, 10x, 20x) Various channels (Phase, Red, Green) Acquire >2000 images per hour Live-cell imaging reagents o Cell Viability o Apoptosis o Proliferation o Phagocytosis

8 Cell-based Assay Development Applications Cell line characterization o Growth curve o Cell viability Early assay development o Time point selection o Specificity Plate effect assessment o Single and bulk preparations Failed sample evaluation o Cell visualization o Time point comparison Cell Line Selection Assay Development Robustness Studies Sample Evaluation

9 P h a s e C o n flu e n c e (% ) Cell Line Characterization Cell Viability + Phase Data Collection Fluorescence Growth Curve Apoptosis Marker T im e (h o u rs )

10 G r e e n C o n flu e n c e (% ) Early Assay Development Time Point Selection Day 1 Day 2 Day D a y 1 D a y 2 D a y L o g [T r e a tm e n t] n g /m L

11 Early Assay Development Specificity Across Three Channels Phase Channel Red Channel: NucLight Red Nuclear-labelled RFP [Proliferation] Green Channel: SYTOX Green Fluorescent Nuclear/Chromosome Counterstain [Apoptosis]

12 Control Treatment A Early Assay Development Phase Cell Viability Apoptosis

13 Control Treatment A Early Assay Development Specificity Phase Analysis Time (h) High Dose Low Dose

14 Early Assay Development Specificity Phase Analysis Treatment A Control A Treatment B Control B

15 Early Assay Development Specificity AUC Phase Analysis

16 Plate Effect Assessment Reference Linearity Preparation Ref Ctrl 50% 75% 100% 150% 175% 50% 75% 100% 150% 175%

17 Plate Effect Assessment Midpoint Concentration Preparation

18 Plate Effect Assessment Low Concentration Preparation

19 Sample Reference Failed Sample Evaluation Cell Visualization Midpoint Concentration Time (h) 0 48 Phase Caspase-3/7

20 u e n c e (% ) Failed Sample Evaluation Time Point Analysis R e fe r e n c e v s S a m p le H R e fe re n c e S a m p le 9 9

21 Conclusions High-content imaging systems provide an in depth understanding of cell-based cytotoxicity assays. Early development can benefit from evaluation of critical factors via imaging. Combination of qualitative and quantitative data provides comprehensive evaluation tools for cellbased assays. Expand understanding of failed samples with more cell-based approach.

22 Acknowledgements Leslie Will Jason Douangpanya Jyoti Velayudhan Thomas Arroll Phil Tsai